The most important trait as a target for crop improvement is yield. Efforts to improve crop yields by developing new plant varieties can be divided into two approaches. One is to reduce crop yield losses by breeding or engineering crop varieties with increased resistance to abiotic stress conditions such as drought, cold, or salt or to biotic stress conditions resulting from pests or disease-causing pathogens. While this approach has value, it does not provide fundamentally improved crop yield in the absence of stress conditions.
The second approach is to breed or engineer new crop varieties in which the basic yield capacity is increased. Classical breeding programs have initially produced substantial gains in improved yield in a variety of crops. A commonly experienced pattern though has been substantial gains in yield initially followed by incremental further improvements that become smaller and more difficult to obtain.
More recently developed approaches based on molecular biology technologies have in principle offered the potential to achieve substantial improvement in crop yield by altering the timing, location, or level of expression of plant genes that play a role in plant growth and/or development. Substantial progress has been made over the past twenty years in identifying plant genes that have a role in plant growth and/or development. Because of the complexity of plant growth regulation and how it relates in the end to yield traits, it is still not obvious which, if any, of these genes would be a clear candidate to improve crop yield.
Much of the work that has been done to identify plant genes with a growth and/or development function has been done in the model plant system Arabidopsis thaliana. One such gene, called REVOLUTA (REV), was originally identified as a loss-of-function mutation in Arabidopsis called rev1 (Talbert et al., Development 121:2723-2735, 1995). This mutation had pleiotropic effects on plant growth and morphology. One phenotype of the rev1 mutation that was of interest was significantly larger seeds. This phenotype was potentially desirable agriculturally but, unfortunately, it was accompanied by undesirable traits such as reduced numbers of flowers and seeds, infertility, and altered leaf morphology. The rev1 mutant exhibits larger leaves, stems, and flowers in addition to larger seeds.
The REV gene was identified by a map based cloning approach (described in WO 01/33944, incorporated herein by reference in its entirety) and it proved to be a transcription factor that belonged to the homeodomain-leucine zipper (HD-ZIP) family of transcription factors. In plants, HD-Zip genes are involved in many developmental pathways, including vascular tissue development, trichome and root hair development, and light-regulated responses. Since rev1 was likely to be a loss of function mutation, efforts were made to knock out REV function in transgenic Arabidopsis by expressing inverted repeat REV (REV-IR) constructs or via co-suppression triggered by strong over expression of the REV gene (WO 01/33944). REV-IR constructs gave a weak rev1 phenotype. The rev1 phenotype including larger, heavier seeds was also observed in REV over expression lines where strong constitutive expression of the REV transgene throughout the plant was driven by the constitutive 35S promoter. Interestingly, this effect correlated with increased REV mRNA levels indicating that it was not due to co-suppression of the endogenous REV gene. The fact that REV over expression and not suppression gave increased seed size was an important finding because it was unanticipated based on the prior work with the rev1 mutant.
Strong constitutive expression of the REV transgene throughout the plant mimicked the large seed phenotype of the rev1 mutant, but it also replicated undesirable phenotypes seen with the rev1 mutant. While the ability to obtain the larger seed size phenotype by the technically straight forward approach of constitutive over expression of a REV transgene showed promise, the undesirable phenotypes meant that this approach would not be viable for commercial agricultural applications.
The agriculturally relevant part of the plant for many crop species is the seed. If the large seed trait could be obtained without the detrimental side effects of general constitutive REV over expression, it would be of potentially great agricultural value. One conceivable approach to overcome this problem was to limit REV over expression specifically to the seed. While many seed-specific promoters are already known and characterized that could potentially be used to drive REV transgene expression, there was no evidence to suggest that, in principle, REV over expression limited to the seed would actually result in increased seed size. The natural function of a plant's endogenous REV gene in the seed is known to be in meristem initiation and adaxial cell fate determination (Otsuga et al., Plant J. 25:223-236, 2001; McConnell and Barton, Development 125:2935-2942 (1998); McConnell et al., Nature 411:709-713, 2001; Emery et al., Curr. Biol. 13:1768-1774, 2003) and not in cell growth or division. Further, it is not known whether the large seed size phenotype in the 35S/REV over expression plants is due to over expression of REV in the seed or in the developing embryo or is instead due to effects on the overall growth and development of the plant caused by REV over expression throughout the plant's tissues.
In addition to the lack of any known biological function for REV in the seed that would have an effect on seed size determination, there is no information to indicate in what part of the seed or during what stage during seed development it might be best to attempt to over express a REV transgene to achieve an increase in seed size.
The present invention provides for the embryo-specific over expression of a growth and/or development related gene during an early stage of embryo development that results in an increase in seed size when compared to a wild-type plant that does not over express the gene. In a particular embodiment the REV gene was overexpressed using an early embryo-specific promoter. The increased seed size trait was achieved without the detrimental side effects seen with constitutive over expression of REV throughout the plant. In addition, embryo-specific over expression of REV gave the unpredicted result of an increase in total seed number per plant when compared to a wild-type plant. The increase in total seed number resulted from an increase in seed number per pod, an increase in the number of racemes per plant, an increase in the number of pods per raceme, a decrease in the rate of seed abortion, or a combination of these effects. Together, the increased seed size and increased seed number resulting from over expression of REV in the developing embryo lead to substantial increases in total yield when compared to a wild-type plant and demonstrates that a gene associated with plant growth and/or development can be overexpressed in association with an early embryo-specific promoter to increase plant yield.